Intraperitoneal administration of adenosine for the treatment of prevention of gastrointestinal of systemic diseases

ABSTRACT

A method for preventing or treating gastrointestinal or systemic diseases in a mammalian subject, comprising: 
     the step of administering a therapeutically effective amount of a composition comprising adenosine or a prodrug thereof into the peritoneal cavity of said subject at a dose that does not achieve pharmacologically active levels in the aortic arterial plasma of said subject.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional ApplicationSerial No. 60/200,360 filed on Apr. 28, 2000.

FIELD OF INVENTION

The present invention relates generally to methods of treatment ofgastrointestinal or systemic diseases. In particular, the presentinvention relates to methods of treatment or prevention ofgastrointestinal or systemic diseases by administration of adenosinewithout causing adverse reactions.

BACKGROUND OF THE INVENTION

Adenosine has promise for the treatment or prevention of several boweldiseases. It is well known that adenosine has the ability to dilate thesplanchnic circulation (see e.g., Proctor, K. G., Circulation Res.59:474 (1986); Proctor, K. G., Circulation Res. 61:187 (1987); Jackson,E. K., Am. J. Physiol. 253:H909 (1987); Kuan, C. J., et al., Am. J.Physiol. 255:H386 (1988); and Holycross, B. J., et al., J. Pharmacol.Exp. Ther. 250:433 (1989) (hereinafter “Holycross, et al. (1989)”), thedisclosures of which are incorporated herein by reference) inhibitplatelet activation (see e.g., Dawicki, D. D., et al., Thromb. Res.43:161 (1986); Paul, S., et al., J. Pharmacol. Exp. Ther. 267:838(1993); and Cristalli, G., et al., Naunyn-Schmiedebergs Archives ofPharmacology 349:644 (1994), the disclosures of which are incorporatedherein by reference) ; and attenuate neutrophil function (see e.g.,Cronstein, B. N., J. App. Phsiol. 76:5 (1994) (hereinafter, “Cronstein(1994)”); Revan, S., et al., J. Biol. Chem. 271:17114 (1996); andJordan, J. E., J. Pharmacol. Exp. Ther. 280:301 (1997), the disclosuresof which are incorporated herein by reference). It has been furtherdemonstrated that locally applied adenosine protects intestinal segmentsfrom ischemia/repercussion injury. See, Kaminski, P. M., et al.,Circulation Res. 65:426 (1989) and Kaminski, P. M., et al., CirculationRes. 71:720 (1992), the disclosures of which are incorporated herein byreference. Adenosine may also be useful in the treatment of inflammatorydiseases of the bowel such as Crohn's disease and ulcerative colitisbecause adenosine inhibits inflammatory cell function (Cronstein(1994)). It is also known that adenosine attenuates the production ofinflammatory cytokines such as TNFα (Wagner, D. R., et al., Circulation97:521 (1998); and Cain, B. S., et al., J. Surg. Res. 76:117 (1998), thedisclosures of which are incorporated herein by reference). Moreover,adenosine inhibits fibroblast proliferation (Dubey, R. K., et al.,Circulation 96:2656 (1997), the disclosure of which is incorporatedherein by reference) extracellular matrix production by fibroblasts(Dubey, R. K., et al., Hypertension 31:943 (1998), the disclosure ofwhich is incorporated herein by reference), and inflammation (Cronstein(1994)), all processes which are involved in the formation of intestinaladhesions (Klein, E. S., et al., J. Surg. Res. 61:473 (1996), thedisclosure of which is incorporated herein by reference). The formationof intestinal adhesions is a major source of surgical morbidity(Thompson, J., Digest. Surg. 15:153 (1998), the disclosure of which isincorporated herein by reference).

The adverse effects of adenosine, however, limit the usefulness of thisagent as a systemically (intravenously or intra-arterially) administereddrug. When so administered, adenosine can cause heart block, asystole,arrhythmias, bradycardia, hypotension, bronchoconstriction and a stressreaction consisting of flushing, headache, dyspnea, chest pressure andnausea. It is therefore unlikely, given the adverse effect profile ofadenosine and adenosine analogues, that systemic administration of theseagents could be used to treat or prevent gastrointestinal diseases ormost systemic diseases. Accordingly, the aim of the present invention isto determine whether adenosine could be administered in such a way thatthe beneficial effects of adenosine therapy may be taken advantage of totreat or prevent gastrointestinal or systemic diseases without attendantadverse effects.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the present invention to providea method of peritoneal lavage with adenosine which providestherapeutically effective levels of adenosine in the intestines of asubject without substantially elevating adenosine levels in the systemiccirculation of the subject.

Another object of the present invention is to provide a method ofperitoneal lavage with an adenosine-releasing “prodrug”, i.e., acompound metabolized to adenosine, to provide pharmacological levels ofadenosine in the intestines of a subject without elevating adenosinelevels in the systemic circulation of the subject.

Still another object of the present invention is to provide a method ofperitoneal lavage with adenosine -5′-monophosphate (“AMP”), anadenosine-releasing prodrug, to provide pharmacological levels ofadenosine in the intestines of a subject without elevating adenosinelevels in the systemic circulation of the subject.

Another object of the present invention is to provide a method ofintraperitoneal administration of adenosine which confers therapeuticbenefits, both locally and systemically, in a subject such as improvedrenal function, improved metabolic status, and improved survival inhemorrhagic shock.

These and other objects of the of the present invention are achieved byone or more of the following embodiments.

In one aspect, the invention features a method for preventing ortreating gastrointestinal or systemic diseases in a mammalian subject,comprising:

the step of administering a therapeutically effective amount of acomposition comprising adenosine or a prodrug thereof into theperitoneal cavity of the subject at a dose that does not achievepharmacologically active levels in the aortic arterial plasma of thesubject.

In preferred embodiments the phosphate ester of adenosine is selectedfrom the group consisting of adenosine-5′-monophosphate,adenosine-5′-diphosphate, adenosine-5′-triphosphate, and adenosine3′:5′-cyclic monophosphate.

In another aspect, the invention features a pharmaceutical compositionfor treating gastrointestinal or inflammatory diseases in a mammaliansubject, wherein the composition comprises adenosine or a prodrugthereof and a pharmaceutically acceptable carrier, and wherein thecomposition is administered into the peritoneal cavity of the subject ata therapeutically effective dose that does not achieve pharmacologicallyactive levels in the aortic arterial plasma of the subject.

In yet another preferred embodiment, the phosphate ester of adenosine isselected from the group consisting of adenosine-5′-monophosphate,adenosine-5′-diphosphate, adenosine-5′-triphosphate, and adenosine3′:5′-cyclic monophosphate.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiment, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show mesenteric blood flow before (Period 1) and during(Periods 2-7) an intramesenteric artery infusion of angiotensin II plusmethoxamine in three groups of rats (n=5 per group). Rats receivedintermittent intraperitoneal lavage with 0.9% saline only (FIG. 1A),increasing concentrations of adenosine (“ADO”) (FIG. 1B), or a fixedhigh concentration of adenosine (FIG. 1C), respectively. Valuesrepresent means±SEM. ^(a)p<0.05 compared with Period 1; ^(b)p<0.05compared with Period 2.

FIGS. 2A-2C show mean arterial blood pressure before (Period 1) andduring (Periods 2-7) an intramesenteric artery infusion of angiotensinII plus methoxamine in three groups of rats (n=5 per group). Ratsreceived intermittent intraperitoneal lavage with 0.9% saline only (FIG.2A), increasing concentrations of adenosine (“ADO”) (FIG. 2B), or afixed high concentration of adenosine (FIG. 2C), respectively. Valuesrepresent means±SEM. ^(a)p<0.05 compared with Period 1; ^(b)p<0.05compared with Period 2.

FIGS. 3A-3C show heart rate before Period 1) and during (Periods 2-7) anintramesenteric artery infusion of angiotension II plus methoxamine inthree groups of rats (n=5 per group). Rats received intermittentintraperitoneal lavage with 0.9% saline only (FIG. 3A), increasingconcentrations of adenosine (“ADO”) (FIG. 3B), or a fixed highconcentration of adenosine (FIG. 3C), respectively. Values representmeans±SEM. ^(a)p<0.05 compared with Period 1; ^(b)p<0.05 compared withPeriod 2.

FIGS. 4A-4D show concentrations of adenosine (FIG. 4A), and itsmetabolites, inosine (FIG. 4B), hypoxanthine (FIG. 4C) and xanthine(FIG. 4D), in the dialysate exiting the microdialysis probe in themesenteric vein and aortic arch during continuous peritoneal lavage withadenosine. Values represent means±SEM (n=5). ^(a)p<0.05 compared withall other points.

FIGS. 5A-5D show concentrations of adenosine (FIG. 5A) and itsmetabolites, inosine (FIG. 5B), hypoxanthine (FIG. 5C), and xanthine(FIG. 5D) in the dialysate exiting the microdialysis probe in themesenteric vein and aortic arch during continuous peritoneal lavage withAMP. Values represent means±SEM (n=5). ^(a)p<0.05 compared with allother points.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term “peritoneal lavage” means placement of asolution into the peritoneal cavity of a subject. Peritoneal lavage maybe carried out once, continuously, or intermittently, in accordance withthe present invention.

“Mesenteric blood flow” refers to the volume of blood per unit timepassing through the mesenteric artery.

“Mean arterial blood pressure” is the average (arithmetic mean) arterialblood pressure over a defined period of time.

A “prodrug of adenosine” means a compound that is metabolized orconverted to adenosine such as, for example, adenosine-5′-monophosphate(“AMP”), adenosine-5′-diphosphate, adenosine-5′-triphosphate, andadenosine 3′:5′-cyclic monophosphate.

“Pharmacologically active” refers to the level of adenosine thatactivates adenosine receptors in a subject.

“Splanchnic circulation” refers to circulation to the abdominal visceraof a subject.

Methods and Results

According to the present invention methods are provided for thetreatment of diseases of the gastrointestinal tract as well as manysystemic diseases. Specifically provided are methods for administeringadenosine or a prodrug thereof to treat or prevent gastrointestinal orsystemic diseases. According to the present invention, the adverseeffects of adenosine that result from systemic administration(intravenous or intraarterial administration) are circumvented byadministering adenosine or a prodrug thereof via single application orintermittent or continuous peritoneal lavage which induce beneficialeffects on the intestines of a subject. This approach can achievepharmacologically active levels of adenosine in the intestinal wall of amammalian or human without producing significant levels of adenosine inthe systemic circulation of the subject. However, there was apossibility that the metabolic barrier to adenosine absorption by thegastrointestinal tract, i.e., intestinal adenosine deaminase (see,Geiger, J. D., et al., Adenosine and Adenine Nucleotides as Regulatorsof Cellular Function, Phillis, J. W., Ed. CRC Press (1991), thedisclosure of which is incorporated herein by reference), would be soeffective in limiting the bioavailability of peritoneally administeredadenosine that active levels of adenosine in the gastrointestinal tractcould only be achieved with concentrations of adenosine in theperitoneal cavity so high that absorption at other sites in theperitoneal cavity would result in overwhelming systemic levels ofadenosine. It was found that according to the present invention,however, that adenosine when administered to a subject by peritoneallavage dilates the splanchnic circulation and increases adenosine levelsin the mesenteric vein, without affecting systemic hemodynamics orincreasing adenosine levels in the arterial circulation. The presentinvention therefore establishes that therapeutically effective levels ofadenosine can be achieved in the peritoneal cavity in a subject withoutattaining pharmacologically active levels in the subject's systemiccirculation.

The methods of the present invention are illustrated in more detailbelow. In one study, rats received an intramesenteric artery infusion ofangiotensin II (30 μg/min) plus methoxamine (3 μg/min) to reducemesenteric blood flow by approximately 60%, and adenosine solutions wereinstilled into the abdominal cavity. In a second study, microdialysisprobes were placed in the mesenteric vein and aortic arch of rats, andthe peritoneal cavity was continuously ravaged with adenosine or AMPsolutions. In a third study, rats were subjected to hemorrhagic shock(2.75 ml of blood removed per 100 grams of body weight over a 15 minuteperiod) for two hours, followed by volume resuscitation for one hour andobservation for 72 hours. Rats received peritoneal lavage with adenosineor vehicle (0.9% saline) beginning 20 minutes after blood withdrawal andcontinuing through the one hour resuscitation period.

As will be seen, peritoneal lavage with adenosine normalized mesentericblood flow (p<0.05) without affecting blood pressure or heart rate.Peritoneal administration of adenosine or AMP induced micromolar levelsof adenosine and inosine in the mesenteric vein (p<0.05), withoutaffecting adenosine or inosine levels in the aorta. Peritoneal lavagewith adenosine increased survival in hemorrhagic shock (9 of 10 animalsin the group treated with intraperitoneal adenosine survived for 72hours, whereas only 4 of 10 animals in the group treated with vehiclesurvived for 72 hours; p<0.05). In rats subjected to hemorrhagic shock,intraperitoneal administration of adenosine significantly (p<0.05)improved metabolic parameters (reduced elevated plasma potassium andplasma lactate and increased arterial plasma pH) and improved renalfunction (reduced elevated blood urea nitrogen levels, an index ofglomerular filtration rate). Therefore, according to the presentinvention, peritoneal lavage with adenosine or one of its prodrugs, AMP,provides pharmacological levels of adenosine in the gastrointestinaltract without systemic side effects. Peritoneal lavage with adenosineimproves survival, metabolic status and renal function in hemorrhagicshock.

Peritoneal lavage with adenosine or adenosine prodrugs according to thepresent invention is useful for the treatment of a number ofgastrointestinal and systemic diseases without the adverse effectsassociated with systemic administration of such drugs. Because adenosineincreases gastrointestinal blood flow, peritoneal lavage with adenosineis useful to treat or prevent gastrointestinal diseases associated withinadequate blood flow to the intestines. Examples of such diseases areocclusion of mesenteric arterial or venous blood vessels caused by, forexample, thrombosis or embolism of the mesenteric arteries or veins,atherosclerosis of the mesenteric arterial blood vessels, necrotizingenterocolitis, intestinal transplantation, traumatic injury to theintestines, or intestinal hypoperfusion due to hemorrhagic shock.Because adenosine is an antiinflammatory substance, peritoneal lavagewith adenosine is useful to treat or prevent gastrointestinal diseasesassociated with inflammation of the bowel. Examples of such diseasesinclude Crohn's disease, ulcerative colitis and reperfusion of the bowelfollowing bowel ischemia for any reason and necrotizing enterocolitis.Because adenosine inhibits the proliferation of fibroblasts and theproduction of collagen by fibroblasts, peritoneal lavage with adenosineis useful to treat or prevent the formation of adhesions in theperitoneal cavity following abdominal surgery. Because adenosineinhibits the activity of various cells in the blood including plateletsand neutrophils and because these blood cells circulate to theintestines, peritoneal lavage with adenosine is useful to treat orprevent systemic diseases in which activation of blood cells,particularly platelets and neutrophils, participate in thepathophysiology of the systemic disease. In this regard, adenosine inthe intestinal circulation inhibits blood cells as they pass through theintestinal vasculature. Even though the adenosine is removed from theintestinal circulation as the blood draining the intestines passesthrough the intestines, liver and lungs, the inhibited blood cellsremains inhibited for a period of time. Examples of diseases treatableor preventable by inhibiting blood cells with peritoneal adenosineinclude hemorrhagic shock, myocardial infarction and stroke.

Although this invention in its preferred embodiments is primarilyaddressed to use in humans, veterinary use is also anticipated and isencompassed by the present invention. In this regard, adenosine or aprodrug thereof may be administered intraperitoneally to dogs, cats,horses, cattle and sheep for gastrointestinal diseases such as, forexample, preventing the formation of adhesions in the peritoneal cavityfollowing surgery.

Adenosine or a prodrug thereof may be admixed with any pharmaceuticallyacceptable carrier or carriers, such as water, saline, physiologicalsalt solutions, Ringer's solution, or any other carrier customarily usedfor intraperitoneal administration to the subject in question.

In that the method of the present invention involves administration ofadenosine or a prodrug thereof intraperitoneally, the drug may besubject to destruction by adenosine deaminase or other enzymes.Therefore adenosine or a prodrug thereof accordingly must beadministered in a larger concentration so that the amount is sufficientto achieve the desired therapeutic effect. The optimal therapeuticconcentration of adenosine or adenosine prodrug in the peritoneal lavagesolution will vary from species to species, individual to individual,and prodrug to prodrug depending on such factors as rate of uptake andmetabolism of adenosine. For example, the rate of adenosine uptake ismuch higher in humans compared with rat red blood cells (Van Belle, H.,Biochim. Biophys. Acta 192:124 (1969) and Jarvis, S. M., et al.,Biochem. J. 208:83 (1982), the disclosures of which are incorporatedherein by reference), and this may necessitate the use of even higherconcentrations of adenosine or adenosine prodrugs in the peritoneallavage solution to produce pharmacologically active concentrations ofadenosine in the intestinal circulation in humans. On the other hand,some individuals may require less adenosine to deliver therapeuticamounts to the intestinal circulation and some prodrugs may be more orless efficient than adenosine in delivering appropriate amounts ofadenosine to the intestinal circulation. It is within the skill of thosein the art to determine the appropriate concentration of adenosine orprodrug thereof to be instilled into the peritoneal cavity of a subject.

The present invention will now be further illustrated by, but is by nomeans limited to, the following examples. It will be apparent to thoseskilled in the art that many modifications, both to materials andmethods may be practical without departing from the purpose and interestof this invention.

In all the examples adult male Sprague-Dawley rats were purchased fromCharles River Laboratories (Wilmington, Mass.) and maintained in theUniversity of Pittsburgh Animal Facility (Pittsburgh, Pa.). Rats wereprovided free access to Prolab Isopro RMH 3000 rodent diet (PMINutrition Intl., Richmond, Ind.) and tap water. Light cycle, relativehumidity and room temperature were 7:00 AM to 7:00 PM, 55% and 22° C.,respectively. Animals weighed 357±11 (mean±SEM) grams at the time ofstudy.

EXAMPLE 1

Rats were anesthetized with Inactin (100 mg/kg, i.p.) (ResearchBiochemicals International, Natick, Mass./Sigma, St. Louis, Mo.) andplaced on a Deltaphase Isothermal Pad (Braintree Scientific, Inc.;Braintree, Mass). Body temperature was monitored with a digital rectalprobe thermometer (Physiotemp Instruments, Inc.; Clifton, N.J.) andmaintained at 37° C. by adjusting a heat lamp above the animal. Thetrachea was cannulated with polyethylene (“PE”)-240 (Becton-Dickinson,Sparks, Md.) to maintain airway patency, a PE-50 catheter was insertedinto the left jugular vein and an intravenous infusion of 0.9% salinewas initiated at 50 μl/min. A left carotid artery catheter (PE-50) wasinserted and was connected to a digital blood pressure analyzer(Micro-Med; Louisville, Ky.) for continuous measurement of mean arterialblood pressure (hereinafter “MABP”) and heart rate. The digital bloodpressure analyzer was set to time-average MABP and heart rate attwo-minute intervals. A transit-time blood flow probe (Model 1RB;Transonic Systems Inc., Ithaca, N.Y.) was placed around the superiormesenteric artery and connected to a transit-time flowmeter (Model T206;Transonic Systems Inc.) to monitor mesenteric blood flow(hereinafter“MBF”) continuously. A 32-gauge needle connected to a PE-1inserted (proximal to the flow probe) into the superior mesentericartery, and an intramesenteric artery infusion of 0.9% saline (50μl/min) was then initiated. The animals were allowed to stabilize forapproximately one hour after the surgical preparation was completed.

The abdominal skin and muscle flaps around the midline incision weresupported in a bowl-shaped fashion to create a basin containing all theviscera, and 40 ml of 0.9% saline prewarmed to 37° C. was instilled intothe peritoneal cavity. All abdominal viscera including the small andlarge intestines, pancreas, stomach, liver and spleen were submergedentirely in the peritoneal lavage fluid. MABP and heart rate weretime-averaged (1100 Hz) over the last six minutes of the first 14-minuteexperimental period,and three readings of MBF were taken at two-minuteintervals over the last six minutes of the first 14-minute experimentalperiod and averaged.

Next, angiotensin II (30 μg/min) (Sigma) plus methoxamine (3 μg/min)(Sigma) were infused into the superior mesenteric artery (50 μl/min).This infusion was maintained for the duration of the experiment. After20 minutes, the peritoneal lavage fluid was withdrawn and replaced withfresh, prewarmed saline. Again, MABP, heart rate and MBF were recordedas described above during the last six minutes of the second 14-minuteexperimental period. At this point, animals were assigned to one ofthree groups. In all groups, five additional back-to-back 14-minuteexperimental periods were conducted in which the peritoneal lavage fluidwas removed and replaced at the beginning of each period and MABP, heartrate and MBF were recorded during the last six minutes of each period.In Group 1 (n=5), only fresh, prewarmed saline was instilled into theperitoneal cavity at the beginning of each experimental period. In Group2 (n=5), increasing concentrations of adenosine (10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M,10⁻⁴ M and 10⁻³ M) dissolved in prewarmed saline were instilled into theperitoneal cavity at the beginning of each experimental period. In Group3, 10⁻³ M adenosine was instilled into the peritoneal cavity at thebeginning of each of the five remaining experimental periods.

Statistical Analysis: Data were analyzed in this example by repeatedmeasures 1-factor analysis of variance. If the analysis of variancedemonstrated a significant effect of the treatments, post-hoccomparisons were calculated with a Fisher's Least Significant Difference(“LSD”) test. Statistical analyses were conducted with NCSS software(Version 6.0; Kaysville, Utah), and the criterion of significance wasp<0.05.

EXAMPLE 2

Rats (n=5) were anesthetized and body temperature monitored andmaintained as described for Example 1. The trachea was cannulated withPE-240 and a PE-50 catheter was inserted into the right jugular vein andan intravenous infusion of 0.9% saline was initiated at 50 μl/min. Afemoral artery catheter (PE-50) was inserted and was connected to adigital blood pressure analyzer for continuous measurement of MABP andheart rate. Next, microdialysis probes (CMA/20 microdialysis probe 4 mm,Bioanalytical Systems, Inc., West Lafayette, Ind.) were inserted intothe aortic arch (via the left carotid artery) and mesenteric vein.Microdialysis probes were perfused with 0.9% saline at 2 μl/min, and thedialysate exiting the probes was collected in a freezing apparatus. Asin Example 1, a basin was constructed from the abdominal wall to createa bowl containing the viscera. However, unlike Example 1, instead ofintermittent lavage with fresh solutions, the abdominal basin wascontinuously perfused with Tyrode's solution (5 ml/min) (Mi, Z., et al.,J. Pharmacol. Exp. Ther. 287:926 (1998), the disclosure of which isincorporated herein by reference) that was maintained at 37° C. by aheated water bath. Thus, the viscera were submerged completely inTyrode's solution that was constantly being replaced. After a 20-minutestabilization period, dialysate from the microdialysis probes wascollected for 20 minutes. Next, the Tyrode's solution perfusing theperitoneal cavity was switched to prewarmed Tyrode's containing 10⁻⁵ Madenosine (Sigma). After 10 minutes, the dialysate was collected foranother 20 minutes. This procedure was repeated two more times as theconcentration of adenosine was increased to 10⁻⁴ M and finally to 10⁻³M. To determine whether AMP would behave as a “prodrug” for the deliveryof adenosine, after a 20 minute washout period, the entire protocol wasrepeated with AMP (10⁻⁵ M, 10⁻⁴ M and 10⁻³ M) (Sigma).

Statistical Analysis: Data were analyzed in this example by repeatedmeasures 1-factor analysis of variance. If the analysis of variancedemonstrated a significant effect of the treatments, post-hoccomparisons were calculated with a Fisher's Least Significant Difference(“LSD”) test. Statistical analyses were conducted with NCSS software(Version 6.0; Kaysville, Utah), and the criterion of significance wasp<0.05.

Sample Analysis: Levels of adenosine and its metabolites, inosine,hypoxanthine and xanthine, in the dialysate samples in this example weremeasured using an Isco (Lincoln, Nebr.) high-pressure liquidchromatographic system (pump model 2350, gradient programmer model 2360,4.6×250-mm C₁₈ column) with UV detection as described by Mi, Z., et al.,J. Pharmacol. Exp. Ther. 273:728 (1995). The chromatography data systemwas JCL 6000 for Windows (Jones Chromatography LTD., Lakewood, Colo.).

EXAMPLE 3

Rats were anesthetized and instrumented with venous and arterialcatheters for blood withdrawal, infusions and monitoring of heart rateand MABP. Hemorrhagic shock was induced by removing 2.75 ml of blood per100 grams of body weight over a 15 minute period. Two hours after bloodwithdrawal, the animals were resuscitated by reinfusing the withdrawnblood and restoring and maintaining a normal blood pressure for one hourby infusing Ringer's solution (Baxter, Deerfield, Ill.) as needed.Beginning 20 minutes after blood withdrawal and continuing for one hourafter reinfusion of the withdrawn blood, rats received either continuousperitoneal lavage with adenosine (10⁻⁴ M) or 0.90% saline.

Results

Example 1: Basal MBFs were similar in the three groups (14.5±2.0,13.8±1.5 and 14.8±1.7 ml/min in Groups 1, 2 and 3, respectively).Intramesenteric artery infusions of angiotensin II (30 ng/min) plusmethoxamine (3 μg/min) caused an immediate decrease in MBF duringexperimental period 2 such that MBFs in the three groups were 6.2±0.8,6.6±1.2 and 6.1±1.0 ml/min in Groups 1, 2 and 3, respectively. Asillustrated in FIG. 1A, in Group 1 (the time-vehicle control group) MBFremained depressed throughout the six experimental periods (periods 2through 7) during the intramesenteric infusions of angiotensin II plusmethoxamine. In Group 2, MBF remained significantly depressed andsimilar in experimental periods 2 through 6 when the adenosineconcentration in the peritoneal lavage fluid was 0, 10⁻⁷ M, 10⁻⁶ M, 10⁻⁵M and 10⁻⁴ M, respectively. However, during the final experimentalperiod (period 7) when the concentration of adenosine in the peritoneallavage fluid was 10⁻³ M (FIG. 1B), MBF increased. In this regard, theMBF during period 7 was significantly greater than the MBF during period2 (intramesenteric artery infusion of angiotensin II plus methoxaminebut no adenosine in the peritoneal lavage fluid) but not significantlydifferent than the MBF during period 1 (no intramesenteric infusion andno adenosine in the peritoneal lavage fluid).

FIG. 1C illustrates the effects of 10⁻³ M adenosine in the peritoneallavage fluid added during experimental periods 3 through 7. This highconcentration of adenosine caused an immediate increase in MBF such thatMBF during experimental periods 3 through 7 were significantly greaterthan MBF during period 2. Also, by period 7, MBF had increased to thepoint that it was no longer significantly different compared withexperimental period 1.

FIGS. 2A-2C and FIGS. 3A-3C summarize the MABP and heart rate,respectively, in the three groups of rats. With regard to MABP, theintramesenteric infusion of angiotensin II and methoxamine caused amarked increase in MABP from period 1 to period 2 (Group 1: 115±5 to146±4 mm Hg; Group 2: 115±4 to 138±6 mm Hg; Group 3: 117±5 to 136±4 mmHg). In all three groups this hypertensive effect of angiotensin II plusmethoxamine was maintained throughout experimental periods 2-7 and wasnot decreased by peritoneal lavage with adenosine. As shown in FIGS.3A-3C in Groups 1 and 2, heart rate was not significantly differentamong the seven experimental periods. In Group 3, angiotensin II plusmethoxamine caused a slight, but significant, decrease in heart rateduring periods 2 and 3; however this effect faded during the subsequentexperimental periods. In Group 3, the high concentration of adenosine inthe peritoneal lavage fluid did not cause bradycardia, the typicalresponse to intravenous adenosine in the anesthetized rat (Holycross etal. (1989)).

Example 2: Basal levels of adenosine, and its metabolites, inosine,hypoxanthine and xanthine, in the dialysate from the mesenteric vein andaorta were less than 0.1 μM. As shown in FIGS. 4A-4D, continuous lavageof the peritoneal cavity with either 10⁻⁵ M, 10⁻⁴ M or 10⁻³ M adenosinedid not significantly alter the levels of adenosine, inosine,hypoxanthine or xanthine in the aorta. Likewise, continuous lavage ofthe peritoneal cavity with either 10⁻⁵ M or 10⁻⁴ M adenosine did notsignificantly alter the levels of adenosine, inosine, hypoxanthine orxanthine in the mesenteric vein. However, continuous lavage of theperitoneal cavity with 10⁻³ M adenosine increased markedly andsignificantly the levels of adenosine (2.17±0.60 μM), inosine (1.03±0.32μM), hypoxanthine (0.18±0.04 μM ) and xanthine (0.26±0.06 μM) in thedialysate exiting the mesenteric vein microdialysis catheter. Likeadenosine, 10⁻³ M AMP also increased purine levels in the mesentericvein, but not in the aorta (FIGS. 5A-5D). The increase in mesentericvenous levels of adenosine with 10⁻³ M AMP tended to be less comparedwith the increase induced by 10⁻³ M adenosine (1.02±0.57 and 2.17±0.60μM, respectively); however, this difference was not statisticallysignificant. Neither adenosine nor AMP significantly affected MABP(113±5 versus 108±4 mm Hg, basal and during 10⁻³ M adenosine,respectively; 112±4 versus 117±7 mm Hg, basal and during 10⁻³ M AMP,respectively) or heart rate (351±15 versus 362±14 beats/min, basal andduring 10⁻³ M adenosine, respectively; 357±17 versus 366±22 beats/min,basal and during 10⁻³ M AMP, respectively).

Example 3: MABP and heart rate were similar between theadenosine-treated and vehicle-treated groups during hemorrhagic shockand resuscitation. Plasma levels of potassium, lactate and blood ureanitrogen were significantly (p<0.05) lower and arterial pH wassignificantly (p<0.05) higher in the adenosine-treated group comparedwith the vehicle-treated group. Survival to 72 hours was significantly(p<0.05) greater in the adenosine-treated group (9 of 10 animals)compared with the vehicle-treated group (4 of 10 animals).

Discussion

The present invention is directed to peritoneal administration ofadenosine to provide therapeutically active levels of adenosine in theintestines without inducing pharmacologically active levels of adenosinein the systemic circulation. The methods were tested in two separateprotocols.

The logic of the first protocol was to induce severe vasoconstriction inthe mesenteric circulation with an intramesenteric artery infusion ofangiotensin II plus methoxamine (α₁-adrenoceptor agonist) so that thedirect vasodilatory effects of adenosine on the splanchnic circulationcould serve as an index of pharmacologically active levels of adenosinein the intestines. Importantly, the intramesenteric artery infusion ofangiotensin II plus methoxamine effectively reduced MBF in all threegroups of animals. Equally important, the effect of angiotensin II plusmethoxamine on MBF was maintained in the time-control animals, thusindicating that any increase in MBF in the adenosine-treated animalscould be interpreted as an effect of the adenosine and not tachyphylaxisto the vasoconstrictors.

In the animals that received intermittent peritoneal lavage withincreasing concentrations of adenosine, low concentrations of adenosinedid not affect MBF. This indicates that the metabolic barrier of theintestines to adenosine bioavailability is large, i.e., it is difficultfor intact molecules of adenosine to be absorbed, avoid metabolism andgain entry into the splanchnic circulation. However, despite the lowbioavailability of intraperitoneal adenosine, when the concentration ofadenosine was increased, active levels of adenosine were achieved in thesplanchnic circulation as evidenced by the marked increase in MBF. Thisfinding was confirmed in the third group of rats in which a highconcentration of adenosine was added to the peritoneal lavage beginningin period 3 and continuing through period 7. In this latter group,adenosine rapidly increased MBF, and MBF continued to increase over theensuing periods.

Since in the anesthetized rat adenosine causes profound bradycardia andhypotension, heart rate and arterial blood pressure were used to monitorfor the presence of pharmacologically active levels of adenosine in thesystem circulation. The intramesenteric artery infusion of angiotensinII plus methoxamine increased MABP, most likely due to spillover of theinfused pressor agents to the systemic circulation. This pressor effectwas fortuitous since it provided an increased MABP upon which to assessadenosine-induced decreases in blood pressure. As with MBF, the systemicpressor response to angiotensin II plus methoxamine was maintained at aconstant level in the time-control animals, thus making any changesobserved in the adenosine-treated animals interpretable.

In Group 2, the group treated with increasing concentrations ofadenosine, adenosine did not cause a reduction in arterial bloodpressure regardless of the concentration instilled into the peritonealcavity. Likewise, when a high concentration of adenosine was instilledinto the peritoneal cavity during period 3 and maintained through period7, MABP was not decreased. In this latter group, there was a very slightand transient increase in MABP during period 4 and period 5 but this wasunlikely due to a direct effect of adenosine on the systemic circulationsince adenosine decreases, rather than increases, arterial bloodpressure in anesthetized rats (Holycross, et al. (1989)). Thus, the MABPdata indicate that even extremely high concentrations of adenosine addedto the peritoneal cavity do not result in pharmacologically activelevels of adenosine in the systemic circulation. This is an importantfinding because it indicates that absorption of adenosine from thesurface of other organs (pancreas, spleen, liver, stomach) and theabdominal walls does not result in systemically active levels ofadenosine despite the presence of extremely high levels of adenosine inthe peritoneal lavage fluid.

The inference from the MABP data that adenosine in the peritoneal lavagefluid does not reach the systemic circulation in any appreciable amountsis supported further by the heart rate data. Since adenosine in theanesthetized rat causes profound bradycardia (Holycross, et al. (1989)),the inability of adenosine in the peritoneal lavage fluid to decreaseheart rate adds weight to the conclusion that adenosine placed in theperitoneal cavity does not reach the systemic circulation in activeamounts.

The first example employed a “bioassay” to test the hypothesis thatperitoneal lavage with adenosine would result in pharmacological levelsof adenosine in the intestines without inducing pharmacological levelsof adenosine in the systemic circulation. However, it is alwayspreferable to address the same question using more than one approach. Inthe second study, microdialysis probes were placed in the mesentericvein and aortic arch to measure more directly whether or not adenosinein the lavage fluid gains access to the splanchnic circulation and/orthe systemic circulation. Microdialysis, rather than direct bloodsampling, was selected for measurement of adenosine levels because ofthe multiple problems of adenosine uptake and metabolism by formedelements in the blood, as well as the problem of ex vivo generation ofadenosine in the sampled blood due to release of ADP from damaged redblood cells (Gewirtz, H., et al., Proc. Soc. Exp. Bio. Med. 185:93(1987), the disclosure of which is incorporated herein by reference).Although absolute levels of purines in the dialysate cannot necessarilybe equated with plasma levels of adenosine, changes in dialysate levelsof purines would reflect changes in plasma levels of purines.

Importantly, no concentration of adenosine in the peritoneal lavagefluid caused any significant increase in adenosine levels or adenosinemetabolite (inosine, hypoxanthine, xanthine) levels in the systemiccirculation. This firmly supports the conclusion from the first examplethat adenosine placed in the peritoneal cavity does not generatepharmacologically active levels of adenosine in the systemiccirculation. Also in line with the first example, high concentrations ofadenosine in the peritoneal cavity cause marked and significantincreases in adenosine and adenosine metabolite levels in the mesentericvein.

Taken together, the data indicate that although the intestinal barrierto adenosine bioavailability is great, this barrier can be overcome withhigh concentrations of adenosine. Moreover, the data indicate thatregardless of the concentration of adenosine in the peritoneal lavagefluid, levels of adenosine in the systemic circulation remain low andbelow the threshold for biological effects. The data indicate thatperitoneally administered adenosine (either by single administration orintermittent lavage or continuous lavage) could be used to treatintestinal diseases. Disease candidates for this method includedisorders leading to bowel ischemic injury, reperfusion injury,inflammation or, adhesion formation. In the present invention, theutility of intraperitoneal administration of adenosine for hemorrhagicshock, a disease characterized by organ (including the gut) ischemicinjury and reperfusion injury is demonstrated. In this regard,peritoneal administration of adenosine improved metabolic status, renalfunction and survival in animals that were subjected to two hours ofhemorrhagic shock.

Peritoneal adenosine also can be used to inhibit circulatinginflammatory cells without systemic side effects. In this regard, bloodflow through the splanchnic circulation would deliver inflammatorycells, such as neutrophils, to the gut where peritoneally administeredadenosine could activate adenosine receptors on the inflammatory cellsto alter their behavior. Although the altered inflammatory cells wouldtravel back to the systemic circulation, the adenosine that altered themwould not. Thus, peritoneally administered adenosine would be useful fordiseases of other organ systems, including the heart, lungs, kidneys andbrain, in which inflammation plays a significant role. This explains inpart the benefit of peritoneally administered adenosine in hemorrhagicshock.

Like adenosine, high concentrations of AMP also significantly increasedadenosine levels in the mesenteric vein without increasing adenosinelevels in the aorta. Since AMP is efficiently metabolized to adenosineby ecto-5′-nucleotidase (an enzyme found widely in the body includingthe gut), AMP functions as a “prodrug” that is readily converted toadenosine. Thus, peritoneal lavage with adenosine prodrugs (examples,AMP, adenosine-5′-diphosphate, adenosine-5′-triphosphate and adenosine3′:5′-cylic monophosphate) can also be used for treating theaforementioned diseases.

A potential concern regarding the intraperitoneal administration ofadenosine is the release of histamine from mast cells, a process thatcan contribute to adenosine-mediated hypotension. However, the inabilityof adenosine in the peritoneal lavage fluid to decrease MABP rules outthe release of significant amounts of systemically bioavailablehistamine from mast cells by intraperitoneal adenosine. Another concernis the effects of adenosine on the kidneys. In this regard, adenosinecauses renal vasoconstriction (Jackson, E. K., Purinergic Approaches InExperimental Therapeutics, Jacobson, K. A., et al., Ed. J. Wiley & Sons,NY. pp. 217-50 (1997), the disclosure of which is incorporated herein byreference). However, the kidneys are retroperitoneal organs, soadenosine applied in the peritoneal cavity would not have access to therenal circulation.

It is unlikely that “chronic” administration of adenosine by peritoneallavage would induce significant toxicity. This hypothesis is based onthree lines of reasoning. First, since systemic concentrations ofadenosine are low during acute peritoneal lavage with highconcentrations of adenosine, it is likely that systemic concentrationsof adenosine also would be low during more prolonged administration ofperitoneal adenosine. This would limit toxicity during chronicadministration of adenosine by the peritoneal route. Second, thedefinition of “chronic” administration in the context of peritoneallavage with adenosine for therapeutic applications would most likely beonly a few days or weeks. This too would limit the likelihood ofsystemic or local toxicity. Third, although the literature is sparsewith regard to chronic administration of adenosine analogues by the oralor peritoneal route, the published articles to date do not mentiontoxicity. In this regard, Adami, M., et al., Eur. J. Pharmacol. 294:383(1985), the disclosure of which is incorporated herein by reference,administered either 2-chloro-N⁶-cyclopentyladenosine (adenosine A₁receptor agonist), 2-hexynyl-5′-N-ethylcarboxamidoadenosine (adenosineA₂ receptor agonist), or 5′-N-ethylcarboamidoadenosine (nonselectiveadenosine receptor agonist) to rats intraperitoneally twice daily for 12days at doses that protected 50% of animals frompentylenetetrazole-induced lethal seizures. Yagil, Y., et al., Am. J.Hyper. 8:509 (1995), the disclosure of which is incorporated herein byreference, administered 6-(O-methylbenzyl) adenosine (adenosine A₂receptor agonist) to rats daily by the oral route for 5 days athypotensive doses. Iwamoto, T., et al., Am. J. Hyper. 7:984 (1994), thedisclosure of which is incorporated herein by reference, gave 2-octynyladenosine (adenosine A₂ receptor agonist) to rats daily by the oralroute for 10 days at hypotensive doses. Finally, Von Lubitz, D. K. J.E., et al., Eur. J. Pharmacol. 249: 271 (1993), the disclosure of whichis incorporated herein by reference, administeredN⁶-cyclopentyladenosine (adenosine A₁ receptor agonist) to miceintraperitoneally once daily for 9 days at doses that increased spatialmemory acquisition in the Morris water maze test. It is important tonote that in the aforementioned studies, metabolically stable adenosineanalogues were used that readily escape adenosine clearance mechanismsin the intestines, liver and lungs. In these studies, pharmacologicallyactive concentrations of adenosine analogues reached the systemiccirculation. Thus, the aforementioned studies differ importantly fromthe present invention in which pharmacologically active levels ofadenosine were not induced by intraperitoneally administered adenosineor adenosine prodrug.

In conclusion, the present invention demonstrates that pharmacologicallyactive concentrations of adenosine can be obtained in the gutcirculation by administering adenosine or AMP into the peritonealcavity. Moreover, it has been established that administration of evenvery high concentrations of adenosine or an adenosine prodrug into theperitoneal cavity does not increase adenosine levels in the systemiccirculation. Thus, intraperitoneal administration of adenosine oradenosine prodrugs will be useful for the treatment of a number ofgastrointestinal or systemic diseases.

Although the invention has been described in detail for the purposes ofillustration, it is to be understood that such detail is solely for thatpurpose and that variations can be made therein by those skilled in theart without departing from the spirit and scope of the invention exceptas it may be limited by the claims.

I claim:
 1. A method for preventing or treating gastrointestinal orsystemic diseases in a mammalian subject, comprising: the step ofadministering a therapeutically effective amount of a compositioncomprising adenosine or a prodrug thereof into the peritoneal cavity ofsaid subject at a dose that does not achieve pharmacologically activelevels in the aortic arterial plasma of said subject.
 2. The method ofclaim 1, wherein said adenosine prodrug is an ester of adenosine.
 3. Themethod of claim 2, wherein said ester of adenosine is a phosphate esterof adenosine.
 4. The method of claim 3, wherein said phosphate ester ofadenosine is selected from the group consisting ofadenosine-5′-monophosphate, adenosine-5′-diphosphate,adenosine-5′-triphosphate, and adenosine 3′:5′-cyclic monophosphate. 5.The method of claim 1, wherein said mammalian subject is a human.
 6. Themethod of claim 1, wherein said disease is formation of adhesions in theperitoneal cavity of said subject during or following abdominal surgery.7. The method of claim 1, wherein said adenosine prodrug is acomposition that releases adenosine.